Conserved Gating Elements in TRPC4 and TRPC5 Channels

TRPC4 and TRPC5 proteins share 65% amino acid sequence identity and form Ca²⁺-permeable nonselective cation channels. They are activated by stimulation of receptors coupled to the phosphoinositide signaling cascade. Replacing a conserved glycine residue within the cytosolic S4–S5 linker of both proteins by a serine residue forces the channels into an open conformation. Expression of the TRPC4_G503S and TRPC5_G504S mutants causes cell death, which could be prevented by buffering the Ca²⁺ of the culture medium. Current-voltage relationships of the TRPC4_G503S and TRPC5_G504S mutant ion channels resemble that of fully activated TRPC4 and TRPC5 wild-type channels, respectively. Modeling the structure of the transmembrane domains and the pore region (S4-S6) of TRPC4 predicts a conserved serine residue within the C-terminal sequence of the predicted S6 helix as a potential interaction site. Introduction of a second mutation (S623A) into TRPC4_G503S suppressed the constitutive activation and partially rescued its function. These results indicate that the S4–S5 linker is a critical constituent of TRPC4/C5 channel gating and that disturbance of its sequence allows channel opening independent of any sensor domain.     

蛋氨酸脑啡肽抑制人胃癌细胞BGC823增殖作用及免疫调节机制

采用不同浓度梯度的蛋氨酸脑啡肽(methionine enkephalin,MENK)体外作用于人胃癌细胞BGC823后,探讨对其增殖影响及其作用机制,为胃癌的免疫治疗提供理论依据。体外培养人胃癌细胞株BGC823,PCR检测阿片受体OGFr的表达;用不同浓度(0、1、2、3、4 mg/mL)的MENK体外作用于BGC823细胞24、48、72、96 h后,MTS检测MENK对其增殖影响;流式细胞术和Annexin V-FITC/PI双染法检测4 mg/mL MENK体外处理48、72 h后BGC823细胞凋亡变化。结果显示,人胃癌BGC823细胞有阿片受体OGFr的表达;MENK可抑制BGC823细胞增殖,且随着剂量的增加和时间的延长,其抑制作用逐渐增强(P0.05);4 mg/mL MENK48 h处理组与空白组相比细胞凋亡率增加,72 h处理组与48 h处理组结果一致(P0.05)。结果表明,MENK可抑制BGC823细胞增殖,具有显著的剂量依赖性和时间依赖性,且可通过诱导细胞凋亡抑制BGC823细胞的增殖。     

MPEP阻断mGlu5抑制肝癌细胞HepG2增殖

代谢型谷氨酸受体5（metabotropic glutamate receptors 5, mGlu 5）在神经系统的多种病理生理过程中发挥重要作用. mGlu5与肝细胞癌的发生发展关系密切,其抑制剂2-甲基-6-(苯乙基）-吡啶（2-methyl-6-(phenylethyl)-pyridine, MPEP）能够促进肝癌细胞凋亡,抑制肝癌细胞的迁移.在此基础上,进一步探讨了MPEP与肝癌细胞增殖之间的关系.结果显示：在无血清和有血清的条件下,MPEP均能显著降低肝癌细胞HepG2的细胞活力.同时发现,有血清条件下MPEP能使HepG2细胞周期停滞在G1 期,显著降低HepG2细胞的DNA合成能力和克隆形成能力,并能下调细胞增殖信号ERK 通路的活性.该研究结果为进一步认识MPEP对肝癌细胞的抑制作用提供了新的实验证据.     

鸟苷酸交换因子P92GEF通过靶向调控RhoA抑制肿瘤细胞增殖侵袭（英文稿）

摘要：Dbl家族鸟苷交换因子（GEFs）是Rho家族蛋白发生恶性转化的主要调控单位,它通过使Rho蛋白从无活性的GDP形式转换为GTP形式的Rho蛋白而发挥作用,参与细胞骨架重排,细胞的生长和活力。P92GEF是一GEFs家族分子。本研究通过Real time PCR对P92GEF在人体48种正常组织中的表达情况进行了测定;GST-pulldown技术对P92GEF的体内GEF活性进行了检测;双荧光素酶报告基因检测技术对下游小分子进行转录因子活性检测;应用免疫荧光双染标记法完成了高表达P92GEF对正常细胞骨架形态的影响;在细胞表型实验中分别使用CCK8法、Transwell法及软琼脂克隆形成实验检测了高表达P92GEF对细胞增殖侵袭迁移及体外成瘤能力的影响。研究结果显示P92GEF有841个氨基酸,具有典型的Dbl家族分子结构域,在肺组织中表达量最高,能够促使正常成纤维细胞中的应力纤维（stress fiber）增多,P92GEF转染的NIH3T3细胞可以独立生长和形成继发性病灶,同时促使细胞增殖,侵袭及克隆形成能力增强,体外转录因子活性检测发现该基因可能与JAK/STAT通路有关。因此,P92GEF是一个典型的鸟苷交换因子家族分子,能激活Rho家族分子RhoA,具有明显的癌基因特征。     

Receptor-mediated regulation of the nonselective cation channels TRPC4 and TRPC5

Mammalian transient receptor potential channels (TRPCs) form a family of Ca(2+)-permeable cation channels currently consisting of seven members, TRPC1-TRPC7. These channels have been proposed to be molecular correlates for capacitative Ca(2+) entry channels. There are only a few studies on the regulation and properties of the subfamily consisting of TRPC4 and TRPC5, and there are contradictory reports concerning the possible role of intracellular Ca(2+) store depletion in channel activation. We therefore investigated the regulatory and biophysical properties of murine TRPC4 and TRPC5 (mTRPC4/5) heterologously expressed in human embryonic kidney cells. Activation of G(q/11)-coupled receptors or receptor tyrosine kinases induced Mn(2+) entry in fura-2-loaded mTRPC4/5-expressing cells. Accordingly, in whole-cell recordings, stimulation of G(q/11)-coupled receptors evoked large, nonselective cation currents, an effect mimicked by infusion of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS). However, depletion of intracellular Ca(2+) stores failed to activate mTRPC4/5. In inside-out patches, single channels with conductances of 42 and 66 picosiemens at -60 mV for mTRPC4 and mTRPC5, respectively, were stimulated by GTPgammaS in a membrane-confined manner. Thus, mTRPC4 and mTRPC5 form nonselective cation channels that integrate signaling pathways from G-protein-coupled receptors and receptor tyrosine kinases independently of store depletion. Furthermore, the biophysical properties of mTRPC4/5 are inconsistent with those of I(CRAC), the most extensively characterized store-operated current.     

LPLUNC1基因对鼻咽癌细胞HNE1生长增殖的抑制作用研究

LPLUNC1在正常的鼻咽组织及人胚鼻咽组织中高表达,而在71%的鼻咽癌中表达下调或缺失,是与鼻咽癌的发生发展密切相关的新基因.通过研究LPLUNC1基因对鼻咽癌细胞系HNE1的影响,进一步确定其与鼻咽癌发生发展的关系.将LPLUNC1基因全长cDNA克隆入pcDNA3.1( )真核表达载体中,通过脂质体介导稳定转染入LPLUNC1低表达鼻咽癌细胞系HNE1中,通过RT-PCR及RNA印迹筛选LPLUNC1高表达的细胞株,并利用细胞生长曲线、MTT、BrdU掺入、流式细胞仪检测、软琼脂集落形成实验及裸鼠成瘤等实验,研究了LPLUNC1对鼻咽癌细胞系HNE1细胞生长、增殖的影响.结果发现,稳定转染LPLUNC1的HNE1细胞的生长速度明显减慢,在MTT与BrdU掺入实验发现LPLUNC1可明显地抑制鼻咽癌细胞的增殖,并且通过流式细胞仪检测也发现,LPLUNC1基因可明显延缓HNE1细胞的细胞周期进程,使G0/G1期细胞增多而S期细胞相对减少.进一步通过软琼脂集落形成及裸鼠成瘤实验发现,LPLUNC1稳定转染后的HNE1细胞集落形成率与集落的大小均小于空白载体细胞,同时能明显地抑制HNE1细胞的体外成瘤.结果表明,LPLUNC1基因能明显抑制鼻咽癌细胞HNE1的生长增殖,是鼻咽癌发生发展中的重要候选抑瘤基因之一.     

5-Aza-C 对鼻咽癌细胞端粒长度及细胞增殖的影响

目的：研究去甲基化药物5-氮杂胞嘧啶核苷（5-Azacytidine,5-Aza-C）对鼻咽癌细胞端粒长度及细胞生长增殖的影响。方法： 常规培养鼻咽癌CNE,CNE1,CNE2 及5-8F细胞系,5-Aza-C 处理鼻咽癌细胞后,甲基化测序聚合酶链反应(MSP)法检测亚端粒 区D4Z4 甲基化,端粒限制性片断检测端粒长度,CCK-8 检测细胞增殖。结果：2.5 uM浓度的5-Aza-C处理后,亚端粒区D4Z4 序 列的甲基化水平明显下降,约在15-20%之间,与处理前的甲基化水平（35-48%）有明显差异,差异均有统计学意义（P < 0.05)。在1 uM和2.5 uM浓度的5-Aza-C 处理后,四种细胞的端粒长度明显缩短,长度在2-4.5kb 之间,差异具有统计学意义(P < 0.05)。5uM 的5-Aza-C处理72 h 后,CNE,CNE1,CNE2 和5-8F 的生存率分别为51.27%,50.46%,48.85%,48.83%,10 uM 的5-azaC 处理72 h 后,CNE,CNE1,CNE2 和5-8F的生存率分别为31.64%,32.34%,30.01%,32.10%,与对照组比较差异有统计学意义（P<0.01）。结 论：5-Aza-C 能缩短端粒长度,抑制鼻咽癌细胞生长增殖活性。     

Corticolimbic expression of TRPC4 and TRPC5 channels in the rodent brain

The canonical transient receptor potential (TRPC) channels are a family of non-selective cation channels that are activated by increases in intracellular Ca(2+) and G(q)/phospholipase C-coupled receptors. We used quantitative real-time PCR, in situ hybridization, immunoblots and patch-clamp recording from several brain regions to examine the expression of the predominant TRPC channels in the rodent brain. Quantitative real-time PCR of the seven TRPC channels in the rodent brain revealed that TRPC4 and TRPC5 channels were the predominant TRPC subtypes in the adult rat brain. In situ hybridization histochemistry and immunoblotting further resolved a dense corticolimbic expression of the TRPC4 and TRPC5 channels. Total protein expression of HIP TRPC4 and 5 proteins increased throughout development and peaked late in adulthood (6-9 weeks). In adults, TRPC4 expression was high throughout the frontal cortex, lateral septum (LS), pyramidal cell layer of the hippocampus (HIP), dentate gyrus (DG), and ventral subiculum (vSUB). TRPC5 was highly expressed in the frontal cortex, pyramidal cell layer of the HIP, DG, and hypothalamus. Detailed examination of frontal cortical layer mRNA expression indicated TRPC4 mRNA is distributed throughout layers 2-6 of the prefrontal cortex (PFC), motor cortex (MCx), and somatosensory cortex (SCx). TRPC5 mRNA expression was concentrated specifically in the deep layers 5/6 and superficial layers 2/3 of the PFC and anterior cingulate. Patch-clamp recording indicated a strong metabotropic glutamate-activated cation current-mediated depolarization that was dependent on intracellular Ca(2+)and inhibited by protein kinase C in brain regions associated with dense TRPC4 or 5 expression and absent in regions lacking TRPC4 and 5 expression. Overall, the dense corticolimbic expression pattern suggests that these Gq/PLC coupled nonselective cation channels may be involved in learning, memory, and goal-directed behaviors.     

Fibroblasts Mobilize Tumor Cell Glycogen to Promote Proliferation and Metastasis

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Sulfatides Inhibit Binding of Helicobacter pylori to the Gastric Cancer Kato III Cell Line

Helicobacter pylori adhere to Kato III and Hela S3 cells in monolayer cultures. To explore whether cell surface glycoconjugates on these two cell lines mediate binding of H. pylori, various carbohydrates, glycoproteins, and glycolipids were tested to inhibit H. pylori cell adhesion. The adhesion was measured (i) with a urease-based assay and (ii) by cells stained with fluorescein. Sodium periodate and sialidase treatment (but not α- or β-galactosidase, heparitinase, lysozyme, or trypsin) inhibited H. pylori binding to both cell lines. Sulfatides and sulfated glycoconjugates (50 μg/ml) but not heparin or a number of simple carbohydrates inhibited binding (1 mg/ml). The two H. pylori strains studied (CCUG 17874 and strain 25) showed high binding of soluble ¹²⁵I-labeled heparin and other sulfated carbohydrate compounds. Received: 5 July 1996 / Accepted: 17 October 1996     

太白银莲花皂苷B对胶质瘤细胞生长抑制的研究

目的:太白银莲花皂苷B(Anemone taipaiensis saponin B)是第一次从太白银莲花中经过系统化学分析和分离鉴定的皂苷之一,所以它的生物学效应目前仍然不清楚。在本研究中,我们首次体外研究太白银莲花皂苷B对胶质瘤细胞系的生物学效应,观察它对胶质瘤细胞增殖的的抑制作用。方法:采用四甲基偶氮唑蓝(MTT)法测定太白银莲花皂苷B对胶质瘤细胞生长曲线的影响,Hoechst 33342细胞核染色后荧光显微镜观察,采用光学显微镜观察细胞的形态学变化。结果:MTT实验结果显示太白银莲花皂苷B对胶质瘤细胞U87MG和U251MG有强烈的生长抑制作用,且具有剂量依赖性,应用SPSS18.0统计软件得出太白银莲花皂苷B对U87MG细胞72 h的抑制浓度为IC25=5.2μmol/L,IC50=6.7μmol/L and IC75=8.7μmol/L,U251细胞的抑制浓度为IC25=6.2μmol/L,IC50=7.9μmol/L and IC75=10.5μmol/L。Hoechst 33342细胞核染色荧光显微镜观察以及光学显微镜下细胞形态观察显示出典型的凋亡细胞形态学特征,经过皂苷B处理后,细胞皱缩成圆球形,细胞核碎裂或者致密浓染,向核膜边缘聚集,染色质浓缩为半月状、车轮状或者马蹄状,凋亡小体出现。这些特征在24 h时更明显。结论:体外实验初步显示,太白银莲花皂苷B对U87MG和U251MG细胞具有明显的增殖抑制作用,并具有促凋亡作用。     

Cytotoxicity to a Tumor Cell Line of Epoxy-Phosphatidylinositol Liposomes

Abstract

Selective cytotoxicity of tumor cells induced by liposomal plant phosphatidylinositol (Ptdlns) has been studied. We could not always obtain cytotoxic plant Ptdlns liposomes in a series of experiments. Moreover, animal Ptdlns occasionally showed cytotoxicity towards tumor cells. By ¹H nuclear magnetic resonance analysis of non-and cytotoxic Ptdlns, it has been suggested that oxidized acyl residues, such as hydroperoxide or dioxetan, may have been present in the cytotoxic Ptdlns. We have prepared epoxy-Ptdlns, as an analogous compound of the oxidized lipid, from noncytotoxic Ptdlns by chemical synthesis. the epoxy-PtdIns liposomes showed cytotoxicity towards tumor cells. In the presence of 100 µM epoxy-Ptdlns liposomes, normal human peripheral lymphocytes survived for 3 days, but Raji human lymphoblastoid-like cells were almost all killed. However, at higher concentrations, epoxy-PtdIns liposomes were also cytotoxic to normal cells.     

沉默Smad4基因表达抑制猪卵巢颗粒细胞增殖并使细胞G_1期阻滞

Smad4是TGF-β/Smad信号通路的核心下游信号分子.为探明Smad4基因对猪卵巢颗粒细胞增殖及细胞周期的影响,采用RNA干扰技术,设计并合成猪Smad4基因的靶向小分子干扰RNA,由LipofectamineTMRNAiMix介导转染体外培养的猪卵巢颗粒细胞.应用实时荧光定量PCR检测Smad4mRNA的干扰效果,应用MTT法、流式细胞术检测细胞增殖和细胞周期的变化,同时应用荧光定量PCR检测转染前后CyclinD1、CyclinB、CyclinA2、CDK1、CDK2、CDK4等周期相关基因的mRNA表达量的变化.实验结果显示,靶向猪Smad4的特异性siRNA序列对Smad4mRNA表达的抑制率为79.85%(P0.01);沉默Smad4可以显著抑制猪卵巢颗粒细胞增殖,并且改变细胞周期分布,G0/G1期细胞比例显著高于各对照组(P0.05),S期细胞比例显著低于各对照组(P0.05),细胞分裂被阻滞;转染36h后CyclinD1、CDK1的mRNA表达量显著低于对照组,CyclinA2、CDK2、CDK4极显著低于对照组,CyclinB差异不显著.综上所述,Smad4是影响猪卵巢颗粒细胞增殖及细胞周期进程的重要基因之一.     

MiR-126 Regulates the ERK Pathway via Targeting KRAS to Inhibit the Glioma Cell Proliferation and Invasion

The activity of some constitutive contained in the extracellular signal-regulated kinase (ERK) pathway plays crucial roles in glioma cell growth and proliferation. Emerging studies have reported that microRNA (miRNA) could regulate the ERK signal pathway by directly targeting various oncogenes. This study enabled us to discover that the average miR-126 expression was significantly decreased in glioblastoma tissues, and this significant decrease was related to high histopathological grades. Our experiment also demonstrated that the over-expression of miR-126 suppressed glioma cell proliferation and invasion in vitro. Kirsten rat sarcoma viral oncogene (KRAS) which is involved in ERK pathway was directly targeted by miR-126 in glioma through binding to two sites in the 3′ untranslated region (3′-UTR) of KRAS mRNA. Notably, the expression level of KRAS was positively correlated to the activity of ERK pathway and its downstream regulators (phosphorylation level of ERK (pERK) and c-Fos). Furthermore, the over-expression of KRAS expression vector without the 3′-UTR partially reverses the tumor-suppressive effects of miR-126. Moreover, the up-regulation of miR-126 contributes to the aberrant activation of the ERK signaling and inhibits cell proliferation and invasion through targeting KRAS. Therefore, it was suspected that miR-126 may be a potential therapeutic target for high-grade glioma.     

TRPM2 Cation Channels Modulate T Cell Effector Functions and Contribute to Autoimmune CNS Inflammation

TRPM2, a highly Ca²⁺-permeable member of the transient receptor potential melastatin-related (TRPM) family of cation channels, is expressed in cells of the immune system. We demonstrate firstly that TRPM2 cation channels on T cells critically influence T cell proliferation and proinflammatory cytokine secretion following polyclonal T cell receptor stimulation. Consistently, trpm2-deficient mice exhibited an attenuated clincal phenotype of experimental autoimmune encephalomyelitis (EAE) with reduced inflammatory and demyelinating spinal cord lesions. Importantly, trmp2-deficient T cells were as susceptible as wildtype T cells to oxidative stress-induced cell death as it occurs in inflammatory CNS lesions. This supports the notion that the attenuated EAE phenotype is mainly due to reduced T cell effector functions but unaffected by potential modulation of T cell survival at the site of inflammation. Our findings suggest TRPM2 cation channels as a potential target for treating autoimmune CNS inflammation.     

RP4 通过miR-183-5p.1上调FOXO1抑制乳腺癌细胞增殖

近年来,越来越多的证据表明,长非编码RNAs在肿瘤发生发展中发挥重要作用。位于12号染色体的长非编码RNA RP4-816N1.7（简称RP4）在乳腺癌细胞中的作用未见报道。我们通过实时荧光定量PCR证实,RP4在乳腺癌细胞中的表达量普遍低于其在正常乳腺上皮细胞MCF-10A中的表达量。RP4在MCF-7和MDA-MB-231中表达量分别比其在MCF-10A中的表达量下调21.57%和91.33%。过表达RP4可明显抑制乳腺癌细胞增殖。敲低RP4可显著增加乳腺癌细胞的增殖能力。生物信息学预测,RP4可能与miR-183-5p.1结合,且叉头蛋白O1(FOXO1)可能是miR-183-5p.1的潜在靶标。实时荧光定量PCR结果提示,RP4可下调miR-183-5p.1,而miR-183-5p.1也可下调RP4和FOXO1的表达。双荧光素酶报告基因结果证实,miR-183-5p.1可与RP4结合,下调其表达,也能与FOXO1 3′UTR结合,抑制其mRNA和蛋白质水平的表达量。最后,本文通过BrdU实验证实,RP4通过FOXO1抑制乳腺癌细胞的增殖。总之,RP4通过内源性结合miR-183-5p.1,上调FOXO1表达,进而抑制乳腺癌细胞增殖。